Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate the bladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degrades fluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studied their fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C). A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320/420 nm, FWHM=50/100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455/525 nm, FWHM=80/50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine’s main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370–430 nm to 395–415 nm would reduce the BWF background by a factor 2.

Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies and rejected 17/20 (85%) noncancerous lesions.

During fluorescence cystoscopy, it is observed that the acquired images are sometimes blurred by a greenish background originating from the bladder washout fluid. Several fluorophores are involved in this overall liquid fluorescence, and their exact origin and relative contributions remain unknown. In this study, the bladder washout fluid is sampled at different times during fluorescence cystoscopy examinations. In total, 32 samples from 12 patients are analyzed with a spectrofluorimeter (excitation range: 350-445 nm, emission range 380-700 nm). This study shows clearly that the
position of the fluorescence peaks (excitation/emission wavelengths: 450/525 nm, 405/625 nm) and shoulder (440/525 nm) is reproducible between different patients. It also suggests that an excitation at wavelengths higher than 400 nm helps to suppress this solution background fluorescence. Additionally, the pH of the solution seems to influence the position of the fluorescence peaks, and this suggests that changing the pH of the examination liquid could help in
avoiding the greenish background.

Hexaminolevulinate fluorescence cystoscopy by the addition of a specific tissue fluorophore enhances the
contrast between benign and malignant epithelial (urothelial) cells in the bladder. Improved detection of flat and
papillary tumors as confirmed in recent phase III clinical trials allows the urologist to obtain a precise
information on the disease extent at the whole surface of the bladder wall. With gaining experience, management
of non muscle invasive bladder cancer improves in readiness and accuracy.

Fluorescence cystoscopy has been recently acknowledged as a useful method to detect early superficial bladder cancer,
even flat lesions. After the instillation of hexaminolevulinic acid (Hexvix) in the bladder for about an hour,
photoactivable porphyrins (PaP), mainly protoporphyrin IX (PpIX) accumulate in the cancerous cells. Although we
observe a selective production of PpIX and an outstanding sensitivity of this method, false positive (FP) lesions
negatively impact its specificity. Carcinogenesis often combines with angiogenesis, and thus changes in vascular
architecture. Therefore, the visualization of the vascular modifications on the fluorescence positive sites is likely to
differentiate false and true positive (TP). New methods including high magnification (HM) cystoscopy are being
investigated by our group, and will yield a reduced number of biopsies and a better characterization of the fluorescence
positive sites. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during
"macroscopic" observation, as well as image acquisition with HM when the endoscope is in contact with the tissue. Each
observed site is biopsied and described by histopathological analysis. The vascular organization (tortuosity, vascular
loops, vascular area and diameter) of the fluorescence positive sites was characterized in parallel with an in situ visual
grading and a dedicated software procedure. We describe here a simple image processing prototype that classifies the
HM images into two classes, according to their pixel distributions. For that purpose, we developed an algorithm in the
image spatial and frequency domain, so that the vascular architecture could be described objectively and quantitatively.

Fluorescence cystoscopy has been recently acknowledged as a useful method to detect early superficial bladder cancer,
even flat lesions. After the instillation of hexaminolevulinic acid (Hexvix®) in the bladder for about an hour,
photoactivable porphyrins (PaP), mainly protoporphyrin IX (PpIX) accumulate in the cancerous cells. Although we
observe a selective production of PpIX and an outstanding sensitivity of this method, false positive (FP) lesions
negatively impact its specificity. Carcinogenesis often combines with angiogenesis, and thus changes in vascular
architecture. Therefore, the visualization of the vascular modifications on the fluorescence positive sites is likely to
differentiate false and true positive (TP). New methods including high magnification (HM) cystoscopy are being
investigated by our group, and will yield a reduced number of biopsies and a better characterization of the fluorescence
positive sites. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during
"macroscopic" observation, as well as image acquisition with HM when the endoscope is in contact with the tissue. Each
observed site is biopsied and described by histopathological analysis. The vascular organization (tortuosity, vascular
loops, vascular area and diameter) of the fluorescence positive sites was characterized in situ. Intrinsic contrast between
the vessels and the tissue was enhanced with an optimization of the spectral design. Preliminary results are presented
here.

Fluorescence detection of early superficial bladder cancer has been well established over the last years. This technique
exploits the selective production and accumulation within cancerous tissues of photoactive porphyrins (PaP), mainly
protoporphyrin IX (PpIX), after the instillation of hexaminolevulinic acid (Hexvix®) in the bladder. Although the
selective production of PpIX and the sensitivity of this procedure are outstanding, its specificity can be improved due to
false positive (FP) lesions. Therefore, our current research focuses on the Characterization of positive sites by high
magnification cystoscopy. Cancerization process often combines with changes in vascular architecture. It is likely that
the visualization of these modifications should allow us to differentiate false and true positive (TP). New methods, using
high magnification (HM) endoscopy, are being investigated by our group, and hopefully resulting in a reduced number
of biopsies. In this study, we are using a dedicated rigid cystoscope, allowing conventional magnification during
"macroscopic" white light and fluorescence observation, as well as image acquisition with HM when the endoscope is in
contact with the tissue. This is realized by an optical setup directly integrated in the cystoscope. We describe here an offclinics
calibration procedure that will allow us to quantify the vessel structure and size once we use this optics to observe
the bladder mucosa.

Our long-term activity in the development of fluorescence imaging for the detection of early superficial bladder cancer
aimed at optimizing the selective production and accumulation of photoactivable porphyrins (PaP), mainly
protoporphyrin IX (PpIX), after the instillation of derivatives of aminolevulinic acid (ALA) within cancerous tissues.
This research eventually led to the approval of hexylaminolevulinate (HAL, Hexvix(R)) in 27 European countries.
Although the selective production of PpIX and the sensitivity of this procedure are outstanding, its specificity is limited
due to false positive lesions that are mainly associated with inflammations of the bladder mucosa. Therefore, our current
research focuses on the improvement of the specificity of this detection method. New methods, using high magnification
(HM) endoscopy, are being investigated by our group in order to discriminate false from true positive findings, and
hopefully resulting in a reduced number of biopsies. In this study, we are using a dedicated magnification cystoscope,
allowing conventional magnification during "macroscopic" white light and fluorescence observation, as well as image
acquisition with HM when the endoscope is in contact with the tissue. This is realized by an optical setup directly
integrated in the cystoscope. The diameter of the field of view of the images is 500 microns in the HM mode and the
resolution is about 3 microns. With this optical setup, our on going study is aimed at observing and characterizing the
neo*-vascularization of the flat fluorescing sites in order to distinguish (pre-)cancerous tissue from inflammation. Thirty
nine biopsies were taken on fluorescence-positive sites. The vascular patterns observed on CIS (n = 7) were significantly
altered in 5 of them (71%), as compared to normal and inflamed mucosa where such alterations were never observed.

This work determines on an in vitro porcine urothelium model the threshold values of different parameters such as photosensitizer concentration, irradiation parameters and production of reactive oxygen species in order to control the damage on normal urothelium and spare about 50% of normal mucosa. For a three hours HAL incubation time, these threshold values were with blue light (0.75J/cm at 75 mW/cm2 or 0.15J/cm2 at 30 mW/cm2) and with white light (0.55J/cm2, at 30 mW/cm2). This means that for identical fluence rates, the threshold value for white light irradiation may be 3 times higher than for blue light irradiation.

Photodynamic therapy is a treatment approach that makes use of a photosensitizer to generate a cell death mechanism in a selective tissue. For its characteristics such as multifocality, high recurrence rate, presence of carcinoma in situ, superficial transitional cell carcinoma of the bladder seems to be an excellent indication for such an approach. Following an overview of thirty years of research, this review focuses on recent developments.

Hexyl aminolevulinate (HAL) fluorescence cystoscopy is being investigated as a new diagnostic tool for the detection of flat urothelial malignancies in bladder cancers. However, the influence of the bladder instillation time on the performance of this detection modality has not been addressed up to now. We report our initial experience comparing different instillation schedules of HAL cystoscopy in the diagnosis of superficial bladder cancer.
A total of 718 fluorescent positive (433) and fluorescence negative (285) biopsies have been taken in the bladder of 143 patients using the Storz D-light fluorescence imaging system (Karl Storz, Tuttlingen, Germany) which allows both white and blue light (380-450 nm) bladder wall inspection.
Following hospitalisation, 50 ml of HAL (8mM) phosphate buffer solution was instilled into the bladder of patients during one hour (1 hour protocol involving 57 patients), or during two hours followed by a two hours resting time after removal of the solution (2+2 hours protocol involving 86 patients). Both instillation subgroups were homogeneous in terms of proportion of high risk disease, previous BCG treatment and/or recurrent disease.
This study indicates that the instillation duration does not influence the results of HAL (Hexvix) fluorescence cystoscopy in our conditions. Compared to the standard use of ALA, HAL (Hexvix) fluorescence cystoscopy allows a significant reduction of the instillation time (to less than one hour) without prejudicing the efficacy of the method, what represents a real advantage in daily clinical practice.

Improvements of endoscopic techniques have renewed the interest of urologists in laser lithotripsy in recent years. Laser energy can be easily transmitted through flexible fibers thereby enabling different surgical procedures such as cutting, coagulating and lithotripsy. The Ho:YAG laser offers multiple medical applications in Urology, among them stone fragmentation. However, the present knowledge of its fragmentation mechanism is incomplete. The objective was therefore to analyze the fragmentation process and to discuss the clinical implications related to the underlying fragmentation mechanism. The stone fragmentation process during Ho:YAG laser lithotripsy was observed by time resolved flash video imaging. Possible acoustic transient occurrence was simultaneously monitored with a PVDF-needle hydrophone. Fragmentation was performed on artificial and cystine kidney stones in water. We observed that though the fragmentation process is accompanied with the formation of a cavitation bubble, cavitation has only a minimal effect on stone fragmentation. Fragment ejection is mainly due to direct laser stone heating leading to vaporization of organic stone constituents and interstitial water. The minimal effect of the cavitation bubble is confirmed by acoustic transients measurements, which reveal weak pressure transients. Stone fragmentation with the Holmium laser is the result of vaporization of interstitial (stone) water and organic stone constituents. It is not due to the acoustic effects of a cavitation bubble or plasma formation. The fragmentation process is strongly related with heat production thereby harboring the risk of undesired thermal damage. Therefore, a solid comprehension of the fragmentation process is needed when using the different clinically available laser types of lithotripsy.

Laser treatment of BPH as minimally invasive therapy has found wide employment in the last few years. The objective here was to study the effects of combined technique of coagulation and vaporization with an Nd:YAG/KTP laser on BPH compared to TURP. Thirty-eight patients presenting symptomatic BPH were randomized and treated either by a laser coagulation/vaporization using an ADD fiber at settings of 40 - 60 W for the Nd:YAG and of 36 W for the KTP alike in 21 cases or by TURP in 17 cases. Symptom score, uroflow and residual urine were assessed preoperatively at 1, 3, 6 and 12 months. No transfusion in any group. Similar postoperative catheterization time. Treatment failure in 2 TURP patients and in 2 laser patients. Comparing AUA score, Qmax and residual urine, both forms of treatment were similar at 1 year. Nd:YAG/KTP laser is equivalent to TURP at 1 year for around 40 g prostates.

The objective of this study was to evaluate and compare the safety and efficacy of the KTP 532 laser to direct vision internal urethrotomy (DVIU) in the management of urethral strictures. A total of 32 patients were randomized prospectively in this study, 14 DVIU and 18 KTP 532 laser. Patients were evaluated postoperatively with flowmetry and in the case of recurrence with cystourethrography at 3, 12, 24 weeks. With the KTP 532 laser complete symptomatic and uredynamic success was achieved in 15 (83%) patients at 12 and 24 weeks. Success rate was lower in the DVIU group with 9 (64%) patients at 12 weeks and 8 (57%) patients at 24 weeks. Mean preoperative peak-flow was improved from 6 cc/s to 20 cc/s at 3, 12 and 24 weeks with the KTP laser. With DVIU mean preoperative peak-flow was improved from 5.5 cc/s to 20 cc/s at 3 weeks followed by a steady decrease to 13 cc/s at 12 weeks and to 12 cc/s 24 weeks. No complication was observed in either group of patients. Our results confirm that stricture vaporization with the KTP 532 laser is a safe and efficient procedure. The better results after laser surgery make it also a valuable alternative in the endoscopic treatment of urethral strictures. These findings suggest a favorable influence of laser surgery on wound healing with less wound contraction and scarring. The lack of contraction of laser wounds might be related to the absence and the lack of organization of myofibroblasts in laser induced lesions.

The prognosis of superficial bladder cancer in terms of recurrence and disease progression is related to the bladder tumor multiplicity and the presence of concomitant 'plane' tumors such as high grade dysplasia and carcinoma in situ (CIS). This study on 33 patients tries to demonstrate the interest of fluorescence cystoscopy in transurethral resection of superficial bladder cancer The method is based on the detection of the protoporphyrin IX (PpIX) induced fluorescence in urothelial cancer cells by topical administration of 5- aminolevulinic acid (ALA). The sensitivity and the specificity of this procedure on apparently normal mucosa in superficial bladder cancer is respectively estimated at 82.9% and 81.3%. Thus, fluorescence cystoscopy is a simple and reliable method in mapping the bladder mucosa, especially in case of multifocal bladder disease and it facilitates the screening of occult dysplasia.

The objective of this study was to evaluate and compare the safety and efficacy of the KTP 532 laser to direct vision internal urethrotomy (DVIU) in the management of urethral strictures. A total of 32 patients were randomized prospectively in this study, 14 DVIU and 18 KTP 532 laser. Patients were evaluated postoperatively with flowmetry and in the case of recurrence with cystourethrography at 3, 12, 24 weeks. With the KTP 532 laser complete symptomatic and urodynamic success was achieved in 15 (83%) patients at 12 and 24 weeks. Success rate was lower in the DVIU group with 9 (64%) patients at 12 weeks and 8 (57%) patients at 24 weeks. With the KTP mean preoperative peak-flow was improved from 6 cc/s to 20 cc/s at 3, 12 and 24 weeks. With DVIU mean preoperative peak-flow was improved from 5.5 cc/s to 20 cc/s at 3 weeks followed by a steady decrease to 13 cc/s at 12 weeks and to 12 cc/s 24 weeks. No complications were observed in either group of patients. Our results confirm that stricture vaporization with the KTP 532 laser is a safe and efficient procedure. It thus represents a valuable alternative in the endoscopic treatment of urethral strictures.

The biomedical use of an optical fiber-based spectro- temporal fluorometer that can endoscopically record the fluorescence decay of an entire spectrum without scanning is presented. The detector consists of a streak camera coupled to a spectrograph. A mode-locked argon ion pumped dye laser or a nitrogen laser-pumped dye laser are used as pulsed excitation light sources. We measured the fluorescence decays of endogenous fluorophores and of ALA-induced- protoporphyrin IX(PPIX) in an excised human bladder with a carcinoma in situ (CIS). Each autofluorescence decay can be decomposed in at least three exponential components for all tissue samples investigated if the excitation is at 425 nm. The decays of the autofluorescence of all normal sites of the human bladder are similar and they differ significantly from the decays measured on the CIS and the necrotic tissue. The fluorescence of the ALA-induced PPIX in the bladder is monoexponential with a lifetime of 15 (plus or minus 1) ns and this fluorescence lifetime does not change significantly between the normal urothelium and the CIS. A photoproduct of ALA-PPIX with a fluorescence maximum at 670 nm and a lifetime of 8 (plus or minus 1) ns was observed. The measurement of the decay of the autofluorescence allowed to correctly identify a normal tissue site that was classified as abnormal by the measurement of the ALA-PPIX fluorescence intensity.

The Nd:YAG/KTP laser offers to the surgeon two wavelengths that can be used to coagulate and vaporize. Our objective was to investigate the combined effect of both wavelengths and to determine the irradiation parameters allowing the largest lesion volume. Chicken breast was irradiated ex vivo. 1064 nm and 532 nm Nd:YAG/KTP laser irradiations were performed sequentially at different combinations with variable fluence and compared to isofluent single wavelengths 40 W irradiation. Although the mean total lesion volume showed no difference between the different wavelengths combinations a significant enhancement of the maximum lesion depth was found under combined irradiation in the 20W/20W conditions. Dual wavelengths irradiation with the Nd:YAG/KTP laser thus induces a specific denaturation process which is more directional and results in an increased total lesion depth. This may represent a ne approach to increase the depth of coagulation necrosis and thus the total lesion volume, thereby improving long term results.

An excellent knowledge of histopathological risk factors of superficial bladder transitional cell carcinoma is mandatory to establish the prognosis of the disease. Presence or absence of carcinoma in situ (CIS) in superficial bladder cancer is one of the most powerful risk indicators. This study examines the usefulness of fluorescence photodetection of neoplastic urothelial foci in bladder cancer following intravesical instillation of delta-aminolevulinic acid (5-ALA). Following bladder instillation of an aqueous solution of 5-ALA in 43 cases, a Krypton ion laser and a Xenon arc-lamp were successively used as excitation source of the PPIX fluorescence. Tissular samples were respectively taken during bladder wall photodetection, either by the means of a video camera, or under direct endoscopic observation. A good correlation was observed between the fluorescence findings and the histopathological diagnosis. On a total of 298 biopsies, 49/110 carcinomatous lesions were detected by the fluorescence, whose more than 36% were CIS. PPIX induced fluorescence with topical bladder instillation of 5-ALA is an efficient and useful method of mapping the mucosa in bladder carcinoma. Moreover, in case of a multifocal disease, this method seems very helpful in finding and treating any residual malignant spots at the end of a transurethral bladder resection.

Clinical results obtained endoscopically with an imaging fluorescence photodetection apparatus based on the use of one excitation wavelength and detection of the fluorescence emission performed in two separate spectral windows are presented. Early cancer photodetection of micro-invasive and in situ carcinoma of the bronchial mucosa after intravenous injection of meso-tetrahydroxyphenylchlorin (m-THPC) was performed during sessions involving photodynamic therapy. Preliminary results are also presented in the context of detecting transitional cell carcinoma of the bladder performed by fluorescence imaging photodetection of protoporphyrin IX (PPIX) following topical application of 5-delta aminolevulinic acid (ALA).

In vivo spectrofluorometric analysis represents a tool to obtain information about fluorophore distribution in tissue. Based on a Peltier-cooled CCD we designed a fluorescence excitation and emission spectrograph which allows to obtain tissue spectra endoscopically and in a clinical environment. Clinical studies were performed on patients with positive cytology or tumor recurrence in the urinary bladder. Patients received a 50 ml instillation of 3% ALA solution at pH 5.5 during 3 to 4 hours and underwent a normal white light cystoscopic examination together with light induced fluorescence photodetection at 5 to 8 hours after the beginning of the instillation. Local fluorescence measurements with a single fiber were performed before photodetection. These showed fluorescence ratios between tumor and normal tissue of 1.5 to 20 with the strongest ratios for exophytic papillary tumors. Fluorescence excitation between 380 nm and 450 nm revealed that the higher Protoporphyrin IX (PPIX) signal on tumor tissue is accompanied by a decrease of the autofluorescence at the emission wavelength of 500 nm.

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